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Novel alpha O-conotoxin peptide GeXIVA new mutant as well as pharmaceutical composition and application thereof

An amino acid, cysteine ​​technology, applied in the fields of pharmacy, biochemistry and molecular biology and neuroscience, which can solve problems such as weak blocking activity

Active Publication Date: 2020-12-01
HAINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The previously discovered α-conotoxins RgIA and Vc1.1 have strong blocking activity on rat α9α10nAChRs, but very weak blocking activity on human α9α10nAChRs, which is 300-400 times lower than that [30]

Method used

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  • Novel alpha O-conotoxin peptide GeXIVA new mutant as well as pharmaceutical composition and application thereof
  • Novel alpha O-conotoxin peptide GeXIVA new mutant as well as pharmaceutical composition and application thereof
  • Novel alpha O-conotoxin peptide GeXIVA new mutant as well as pharmaceutical composition and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0155] Example 1: Sequence design and artificial synthesis of a new mutant of αO-conotoxin GeXIVA

[0156] On the basis of the amino acid sequence of αO-conotoxin GeXIVA ( figure 1 , SEQ ID NOs: 1 and 25 in Table 1-2), the inventors creatively designed a series of new polypeptide mutants, the amino acid sequence of which is as SEQ ID NOs: 2-24, 25 in Table 1-7 -117 shown.

[0157] The linear peptides of the polypeptides listed in Table 1-7 were artificially synthesized by the Fmoc method. The specific method is as follows:

[0158] The resin peptide is artificially synthesized by Fmoc chemical method, and the resin peptide can be synthesized by a peptide synthesizer or manual synthesis. Except for cysteine, the remaining amino acids use standard side chain protecting groups. For a polypeptide containing 4 cysteines (Cys), if its disulfide linkage is [C1-C4, C2-C3], the -SH of the second and third cysteines (Cys) Protected with Trt (S-trityl), the -SH of the 1st and 4th ...

Embodiment 2

[0189] Example 2: A method for detecting the receptor binding activity of αO-conotoxin GeXIVA and its mutants.

[0190] References [27，37]The method in, and the instructions of the in vitro transcription kit (mMessage mMachine in vitrotranscription kit (Ambion, Austin, TX)) prepared various rat neurotype nAChRs subtypes (α3β4, α6 / α3β4, α9α10, α4β2, α4β4, α3β4, α2β2, α2β4, α7), human α9α10, and mouse muscle nAChRs (α1β1δε) cRNA, the concentration was measured by OD value under UV 260nm. Xenopus laveis oocytes (frog eggs) were dissected and collected, and cRNA was injected into the frog eggs, and the injected amount of each subunit was 5-10 ng cRNA. Frog eggs were cultured in ND-96. cRNA was injected within 1-2 days after frog egg collection, and used for voltage-clamp recordings of nAChRs within 1-4 days after injection.

[0191] One cRNA-injected frog egg was placed in a 30 μL Sylgard recording tank (diameter 4 mm × depth 2 mm), and ND96 perfusate (96.0 mM NaCl, 2.0 mM KC...

Embodiment 3

[0194] Example 3: αO-conotoxin GeXIVA[1,2] and its alanine scanning mutants are different subtypes of nAChRs blocking activity

[0195] αO-conotoxin GeXIVA[1,2] (SEQ ID NO:1, figure 1 ) and its alanine-scanning mutants (SEQ IDNOs:2-24) (Table 1) for the blocking activity of rat neurotype α9α10, α7, and mouse muscle type α1β1δεnAChRs, which are three very similar receptor subtypes , the half-blocking dose (IC 50 ) are summarized in Table 8. The ratios between the blocking activities of these mutants against the 3 subtypes are also summarized in Table 8.

[0196] Table 8: Effects of αO-conotoxin GeXIVA[1,2] (SEQ ID NO:1) and its alanine scanning mutants (SEQ IDNOs:2-24) on rat neurotype α9α10, α7, and mouse muscles Blocking activity of type α1β1δε nAChRs

[0197]

[0198] a Numbers outside brackets refer to the half-blocking dose (IC 50 ), the unit is nanomole (nM); the data in brackets is the half-blocking dose of 95% confidence interval (IC 50 ) range.

[0199] ...

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Abstract

The invention belongs to the field of biochemistry, molecular biology and neurosciences, and relates to a novel alpha O-conotoxin GeXIVA mutant and a pharmaceutical composition and application thereof. Specifically, the invention relates to a separated polypeptide. The amino acid sequence of the polypeptide is obtained by removing one or more amino acids in a mother sequence or replacing one or more amino acids in the mother sequence with the same number of L-type amino acids or D-type amino acids, wherein the mother sequence is SEQ ID NO:1, SEQ ID NO:62 or SEQ ID NO:63; and the polypeptide has an activity of blocking alpha9alpha10nAChR. The polypeptide disclosed by the invention can specifically block the alpha9alpha10nAChR, and has a good application prospect in the aspects of preparinganalgesic drugs, drugs for treating related mental diseases and cancers, neuroscience tool drugs and the like.

Description

technical field [0001] The invention belongs to the fields of pharmacy, biochemistry, molecular biology and neuroscience, and relates to αO-conotoxin peptide GeXIVA and a series of new mutants thereof, its pharmaceutical composition, its preparation method and its application. The present invention also relates to a method for blocking nicotinic acetylcholine receptors (nAChRs), and the pharmaceutical use of the conotoxin peptide. Background technique [0002] A variety of polypeptide toxins produced in the venom of carnivorous molluscs living in tropical oceans are called conotoxins or conopeptides. Conotoxins have the special function of specifically binding to various ion channels and receptors in animals [1] , has leapt to the first place in the study of animal toxins, and is known as the "treasure house of marine drugs" and "the untapped treasury". Conotoxins have become a hot spot in neuroscience research and new drug development. [0003] Conotoxins have diverse th...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K19/00C12N15/12A61K38/17A61P25/00A61P35/00A61P29/00A61P33/00
CPCC07K14/43504A61P25/00A61P35/00A61P29/00A61P33/00C07K2319/00A61K38/00A61K38/17C07K14/435C07K19/00A61P17/02A61P25/24A61P25/28A61P25/04A61P25/16A61P25/18C12N15/63
Inventor 罗素兰长孙东亭朱晓鹏吴勇
Owner HAINAN UNIVERSITY
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